Políticas contables significativas (continuación)

In a model organism E. coli, chemoreceptors form mixed clusters and subsequently large arrays that localize to the cell poles along with the CheA histidine kinase and the CheW coupling protein as revealed by PALM (photoactivated localization microscopy), fluorescence microscopy, and cross-linking studies (Greenfield et al., 2009; Kentner, 2006; Studdert and Parkinson, 2003). Cryo-ET studies of 13 distinct bacterial species showed that such architecture is universally conserved and likely contributes to signal gain and amplification (Briegel et al., 2009). Interaction of chemoreceptors with the histidine kinase CheA is required for chemotaxis signaling but it appears not to be required for chemoreceptor cluster formation (Kentner et al., 2006). In the absence of CheA in E. coli, chemoreceptors formed multiple lateral clusters and appeared diffused. In the absence of the coupling protein CheW, chemoreceptors clusters were localized at the cell poles,, but these clusters appeared less compact compared to the wild type clusters (Kentner et al., 2006). Recent cryo-ET studies in E. coli confirmed that CheA and CheW proteins are not required for chemoreceptors cluster formation but that these proteins are essential for the formation of large receptor arrays found at the cell poles (Briegel et al., 2014). Since more than half of the bacterial species which genomes have been sequenced, contain more than one CheA homologue, how multiple CheAs and numerous receptors organize within the cells and how the presence of these multiple proteins affect the formation of chemoreceptors clusters.

The genome of the alphaproteobacterium A. brasilense encodes for 41 chemoreceptors, and several CheA and CheW homologs (Wisniewsky-Dye et al., 2012). Experiments performed to date indicate that che1 and che4 operons contribute to chemotaxis via an effect on swimming

51 speed (Bible et al., 2008) and reversal frequency (Kumar, 2012; unpublished data). Additional data also hint at a potential signaling cross-talk during chemotaxis, which may be initiated at the receptors level (Stephens et al., 2006; Russell et al., 2013). Experimental evidence also suggest that several chemoreceptors in A. brasilense (Tlp1 and AerC) signal via both Che1 and Che4 (Xie et al., 2010; Bible et al., 2012; Russell et al., 2013). Since A. brasilense has at least two CheAs interacting with chemoreceptors (CheA1 and CheA4) their effect on chemoreceptors cluster formation and localization is expected to be different from that described in E. coli. The observations made in A. brasilense and described above suggest several possibilities that are not mutually exclusive regarding the organization of chemoreceptor arrays within this bacterial species. One possibility is that distinct arrays cluster mixed sets of chemoreceptors, with one cluster dedicated to relay Che1 signals and another for Che4 signaling. Another possibility is that chemoreceptors are organized in a single large array that also interacts with both Che1 and Che4 proteins. Under both possibilities, the organization of chemoreceptors within the arrays and their interaction with Che1 and Che4 protein must be distinct to account for signal integration in chemotaxis and cross-talk signaling. In this study we elucidated organization of bacterial chemoreceptors in respect to one another and to chemotaxis proteins from Che1 and Che4 pathways using fluorescence microscopy and in vivo BACTH assay.

Until this study, the subcellular localization and organization of chemoreceptors from A. brasilense that belong to 5 different signaling domain classes was unknown. Based on the results of this study, we propose that Tlp1 and Tlp4a chemoreceptors belong to two distinct clusters (Figure 18). This is based on the fact that removal of Tlp1 does not affect Tlp4a localization, expression, and cluster formation. In addition, Tlp1 and Tlp4a were not found to interact in vivo.

52 Finally, recent reports demonstrate that receptors from different signaling domain classes in E. coli (40H and 36H) do not intermixed in clusters (Herrera Seitz et al., 2014). Tlp1 (and 32 other chemoreceptors) belongs to the 38H class, while Tlp4a belongs to the 36H class; therefore, we propose that these two receptors (and other receptors from the same class) do not form mixed clusters and in fact likely belong to two physically distinct clusters.

Through fluorescence microscopy studies, we have found that chemoreceptors polar localization and recruitment to the clusters depends on the presence of CheA4. CheA4 was also found to be interacting with the receptors and both CheW1 and CheW4 in vivo. The later observation if significant because it provides a mechanistic rationale for the observed signaling cross-talk between the Che1 and Che4 pathways. CheA1 strongly interacted with AerC but it was not found to interact with Tlp1 or Tlp4a. Based on these evidence, we are proposing the following model for chemoreceptors clusters organization in A. brasilense. Cluster 1 is comprised of Tlp4a and the other two chemoreceptors belonging to the same signaling class, and it interacts with CheA4 and CheW4. Indeed we have found that Tlp4a weakly interacted, in vivo, with CheA4 and CheW4 (Figure 18). Tlp4a did not interact with CheW1, and could therefore be coupled to CheA4 only indirectly, likely through CheW4. CheA4 interacted with both CheW1 and CheW4; therefore, in this particular cluster, CheA4 may be coupled to the other receptors through CheW1. Since Tlp4a did not interact with itself in vivo (and likely does not form dimers), we are proposing that another chemoreceptor of the same signaling class dimerizes with Tlp4a to form heterodimers and thus permits signaling and cluster assembly. AerC was found to strongly interact with Tlp4a, and Tlp4a’s cluster architecture was affected in the ΔaerC

53 background. Therefore, we propose that AerC is also present in Cluster 1 (on the cytoplasmic side).

The second cluster is comprised of the chemoreceptors belonging to the 38H class (such as Tlp1 and Tlp2) and interact with both CheA1 and CheA4. This is supported by the fact that polar localization of Tlp1 depends on the presence of CheA4, and that CheA4 and Tlp1 strongly interacted in vivo. Tlp1 did not interact with CheA1 or CheW1 in vivo, but it was found to signal via the Che1 pathway (Russell et al., 2013). In addition, CheA1 and CheA4 were found to strongly interact in vivo and may form heterodimers. Therefore, we propose that Tlp1 relays signal through Che1 via interaction with CheA4-CheA1 heterodimers. Another possibility is that CheA1 may be present in Cluster 2 but does not physically interact with Tlp1 (Figure 18B). AerC was found to interact with Tlp1, CheA1, and CheW1 in vivo and to affect the localization of Tlp1 and Tlp2. AerC has also been previously shown to localize to the cell poles in a Che1- dependent manner (Xie et al., 2010). Therefore, we hypothesize that this soluble chemoreceptor is present in Cluster 2. Our fluorescent microscopy results suggest that soluble chemoreceptor AerC has an effect on transmembrane chemoreceptor clustering since the absence of AerC affected localization of Tlp1/Tlp2 as well as the architecture of the Tlp4a cluster. To our knowledge, a similar effect of a soluble receptor on the localization of transmembrane chemoreceptors has not been reported.

In conclusion, our study reveals a novel mode of bacterial chemoreceptor organization in which transmembrane chemoreceptors form two distinct clusters that preferentially interact with one CheA (cluster 1) or both CheA1 and CheA4 (cluster 2) and in which a soluble chemoreceptor AerC plays a structural role in transmembrane chemoreceptor clustering.

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VITA

Anastasia Aksenova (De Cerqueira) was born in Kaluga, Russia. She attended 17th Elementary, Middle, and High School which she graduated in 2001. She continued her education in French-Russian Institute of Business Administration where she received Bachelor’s Degree in Business Administration. In 2005, Anastasia switched her professional interests to science. She moved to Knoxville, TN in June 2005 where she attended Pellissippi State Technical Community College. She graduated with Associate’s Degree in Biology, and then got her Bachelor’s Degree in Biochemistry from Maryville College (Maryville, TN) in 2011. She participated in a Summer Internship Program at Oak Ridge National Laboratory in 2008 that made her interested in a career in biological sciences. Anastasia was offered a graduate teaching position at the University of Tennessee in Knoxville in 2011 where she worked under Dr. Gladys Alexandre. She completed her Master’s of Science Degree in Biochemistry, Cellular and Molecular Biology in 2014.